The present invention relates to methods and devices for the removal of substances from blood products and particularly to methods and devices for the removal of psoralens and psoralen photoproducts from plasma that contains platelets without significantly affecting platelet function.
Pathogen contamination within the blood supply remains an important medical problem throughout the world. Although improved testing methods for hepatitis B (HBV), hepatitis C (HCV), and HIV have markedly reduced the incidence of transfusion associated diseases, the public is losing trust in the safety of the blood supply due to publicity of cases of transfusion related transmission of these viruses.
For example, the recent introduction of a blood test for HCV will reduce transmission of this virus; however, it has a sensitivity of only 67% for detection of probable infectious blood units. HCV is responsible for 90% of transfusion associated hepatitis. Melnick, J. L., Abstracts of Virological Safety Aspects of Plasma, Cannes, Nov. 3-6, 1992 (page 9). It is estimated that, with the test in place, the risk of infection is 1 out of 3300 units transfused.
Similarly, while more sensitive seriological assays are in place for HIV-1 and HBV, these agents can nonetheless bed transmitted by seronegative blood donors. International Forumn: Vox Sang 32:346 (1977). Ward, J. W., et al., N. Engl. J. Med., 318:473 (1988). Up to 10% of total transfusion-related hepatitis and 25% of severe icteric cases are due to the HBV transmitted by hepatitis B surface antigen (HBasAg) negative donors. To date, fifteen cases of transfusion-associated HIV infections have been reported by the Center for Disease Control (CDC) among recipients of blood pre tested negative for antibody to HIV-1.
Furthermore, other viral, bacterial, and agents are not routinely tested for, and remain a potential threat to transfusion safety. Schmunis, G. A., Transfusion 31:547-557 (1992). In addition, testing will not insure the safety of the blood supply against future unknown pathogens that may enter the donor population resulting in transfusion associated transmission before sensitive tests can be implemented.
Even if seroconversion tests were a sufficient screen, they may not be practical in application. For example, CMV (a herpes virus) and parvo B19 virus in humans are common. When they occur in healthy, immunocompetent adults, they nearly always result in asymptomatic seroconversion. Because such a large part of the population is seropositive, exclusion of positive units would result in substantial limitation of the blood supply.
An alternative approach to eliminate transmission of viral diseases through blood products is to develop a means to inactivate pathogens in transfusion products. Development of an effective technology to inactivate infectious pathogens in blood products offers the potential to improve the safety of the blood supply, and perhaps to slow the introduction of new tests, such as the recently introduced HIV-2 test, for low frequency pathogens. Ultimately, decontamination technology could significantly reduce the cost of blood products and increase the availability of scarce blood products.
To be useful, such an inactivation method i) must not adversely affect the function for which the blood product is transfused, ii) must thoroughly inactivate existing pathogens in the blood product, and iii) must not adversely affect the recipients of the blood product. Several methods have been reported for the inactivation or elimination of viral agents in erythrocyte-free blood products. However, most of these techniques are completely incompatible with maintenance of the function of platelets, an important blood product. Examples of these techniques are: heat (Hilfenhous, J., et al., J. Biol. Std. 70:589 (1987)), solvent/detergent treatment (Horowitz, B., et al., Transfusion 25:516 (1985)), gamma-irradiation (Moroff, G., et al., Transfusion 26:453 (1986)), UV radiation combined with beta propriolactone, (Prince A. M., et al., Reviews of Infect. Diseases 5:92-107 (1983)), visible laser light in combination with hematoporphyrins (Matthews J. L., et al., Transfusion 28:81-83 (1988); North J., et al., Transfusion 32:121-128 (1992)), use of the photoactive dyes aluminum phthalocyananine and merocyanine 540 (Sieber F., et al., Blood 73:345-350 (1989); Rywkin S., et al., Blood 78(Suppl 1):352a (Abstract) (1991)) or UV alone (Proudouz, K. N., et al., Blood 70:589 (1987)).
Other methods inactivate viral agents by treatment with furocoumarins, such as psoralens, in the presence of ultra-violet light. Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985); Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to a psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a second photon can lead to formation of a diadduct which functions as an interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058 (1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst et al., U.S. Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby incorporated by reference.
The covalently bonded psoralens act as inhibitors of DNA replication and thus have the potential to stop the replication process. Due to this DNA binding capability, psoralens are of particular interest in relation to solving the problems inherent in creating and maintaining a pathogen-free blood supply. Some known psoralens have been shown to inactivate viruses in some blood products. H. J. Alter et al., The Lancet (ii:1446) (1988); L. Lin et. al., Blood 74:517 (1989) (decontaminating platelet concentrates); G. P. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, hereby incorporated by reference, describe the use of a combination of 8-methoxypsoralen (8-MOP) and irradiation. P. Morel et al., Blood Cells 18:27 (1992) show that 300 xcexcg/mL of 8-MOP together with ten hours of irradiation with ultraviolet light can effectively inactivate viruses in human serum. Similar studies using 8-MOP and aminomethyltrimethyl psoralen (AMT) have been reported by other investigators. Dodd RY, et al., Transfusion 31:483-490 (1991); Margolis-Nunno, H., et al., Thromb Haemostas 65:1162 (Abstract)(1991). Indeed, the photoinactivation of a broad spectrum of microorganisms has been established, including HBV, HCV, and HIV, under conditions different from those used in the present invention and using previously known psoralen derivatives. [Hanson, C. V., Blood Cells, 18:7-24 (1992); Alter, H. J., et al., The Lancet ii:1446 (1988); Margolis-Nunno, H. et al., Thromb Haemostas 65:1162 (Abstract) (1991).]
Psoralen photoinactivation is only feasible if the ability of the psoralen to inactivate viruses is sufficient to ensure a safety margin in which complete inactivation will occur. On the other hand, the psoralen must not be such that it will cause damage to blood products. The methods just described, when applied using known psoralens, require the use of difficult and expensive procedures to avoid causing damage to blood products. For example, some compounds and protocols have necessitated the removal of molecular oxygen from the reaction before exposure to light, to prevent damage to blood products from oxygen radicals produced during irradiation. See L. Lin et al., Blood 74:517 (1989); U.S. Pat. No. 4,727,027, to Wiesehahn. This is a costly and time consuming procedure.
Finally, some commonly known compounds used in photochemical decontamination (PCD) exhibit undesirable mutagenicity which appears to increase with increased ability to kill virus. In other words, the more effective the known compounds are at inactivating viruses, the more injurious the compounds are to the recipient, and thus, the less useful they are at any point in an inactivation system of products for in vivo use.
A new psoralen compound is needed which displays improved ability to inactivate pathogens and low mutagenicity, without causing significant damage to blood products for which it is used, and without the need to remove oxygen, thereby ensuring safe and complete inactivation of pathogens in blood decontamination methods. In addition, a device is needed that is capable of removing from blood products both residual levels of and photoproducts created by a suitable psoralen, thereby allowing efficient and economical widespread use of PCD treatment of such blood products.
The present invention provides new psoralens and methods of synthesis of new psoralens having enhanced ability to inactivate pathogens in the presence of ultraviolet light which is not linked to mutagenicity. The present invention also provides methods of using new and known compounds to inactivate pathogens in health related products to be used in vivo and in vitro, and particularly, in blood products and blood products in synthetic media.
The present invention contemplates a method of inactivating pathogens in a platelet preparation comprising, in the following order: a) providing, in any order, i) a synthetic media comprising a compound selected from the group consisting of 4xe2x80x2-primaryamino-substituted psoralens and 5xe2x80x2-primaryamino-substituted psoralens; ii) photoactivating means for photoactivating said compound; and iii) a platelet preparation suspected of being contaminated with a pathogen having nucleic acid; b) adding said synthetic media to said platelet preparation; and c) photoactivating said compound so as to prevent the replication of substantially all of said pathogen nucleic acid, without significantly altering the biological activity of said platelet preparation. The pathogen may be a virus, or a bacteria. Its nucleic acid may be single stranded or double stranded, DNA or RNA. The photoactivating means comprises a photoactivation device capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm. The intensity may be between 1 and 30 mW/cm2 and the platelet preparation is exposed to said intensity for between 1 second and thirty minutes. The spectrum of electromagnetic radiation may be wavelengths between 320 nm and 380 nm.
In one embodiment the compound displays low mutagenicity. It may be added to said platelet preparation at a concentration of between 0.1 and 250 xcexcM. And the method may be performed without limiting the concentration of molecular oxygen.
The 4xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 4xe2x80x2 carbon atom, selected from the group comprising:
(CH2)uxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2; and
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2R3, and R4 are independently selected from the group comprising O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents R5, R6, and R7 on the 4, 5xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof
Alternatively, the 5xe2x80x2-primaryamino-substituted psoralen comprises: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
(CH2)uxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2; and
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4, 4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5, and where when R1 is selected from the group comprising xe2x80x94(CH2)uxe2x80x94NH2, R6 is H; or a salt thereof
Finally, the 5xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
(CH2)uxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2; and
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 3 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4, 4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
In one embodiment, at least two compounds are present. In another embodiment, the synthetic media further comprises sodium acetate, potassium chloride, sodium chloride, sodium citrate, sodium phosphate and magnesium chloride, and may also include mannitol and/or glucose.
In one embodiment, the synthetic media is contained in a first blood bag and said platelet preparation is contained in a second blood bag, the synthetic media being added to the platelet preparation in step (b) by expressing the synthetic media from the first blood bag into the second blood bag via a sterile connection.
In a preferred embodiment, the compound is either 5xe2x80x2-(4-amino-2-oxa)butyl-4,4xe2x80x2,8-trimethylpsoralen or 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen.
In one embodiment, the method described above includes administering said platelet preparation by intravenous infusion to a mammal.
The present invention contemplates a method of inactivating pathogens in a platelet preparation comprising, in the following order: a) providing, in any order, i) a synthetic media comprising a buffered saline solution and a compound displaying low mutagenicity, selected from the group consisting of 4xe2x80x2-primaryamino-substituted psoralens and 5xe2x80x2-primaryamino-substituted psoralens, contained in a first blood bag; ii) photoactivating means for photoactivating said compound; and iii) a platelet preparation suspected of being contaminated with a pathogen having nucleic acid, contained in a second blood bag; b) adding said synthetic media to said platelet preparation by expressing said synthetic media from said first blood bag into said second blood bag via sterile connection means; and c) photoactivating said compound so as to prevent the replication of substantially all of said pathogen nucleic acid, without significantly altering the biological activity of said platelet preparation. The pathogen may be a virus or a bacteria. Its nucleic acid may be single stranded or double stranded, DNA or RNA. The photoactivating means comprises a photoactivation device capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm. The intensity may be between 1 and 30 mW/cm2 and the platelet preparation is exposed to said intensity for between 1 second and thirty minutes. The spectrum of electromagnetic radiation may be wavelengths between 320 nm and 380 nm.
In one embodiment the compound displays low mutagenicity. It may be added to said platelet preparation at a concentration of between 0.1 and 250 xcexcM. And the method may be performed without limiting the concentration of molecular oxygen.
The 4xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 4xe2x80x2 carbon atom, selected from the group comprising:
(CH2)uxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2; and
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents R5, R6, and R7 on the 4, 5xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
Alternatively, the 5xe2x80x2-primaryamnino-substituted psoralen comprises: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
(CH2)uxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2; and
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yR4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4, 4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5, and where when R1 is selected from the group comprising xe2x80x94(CH2)uxe2x80x94NH2, R6 is H; or a salt thereof
Finally, the 5xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
(CH2)uxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2;
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2; and
(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yR4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 3 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4, 4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
In one embodiment, at least two compounds are present. In another embodiment, the synthetic media further comprises sodium acetate, potassium chloride, sodium chloride, sodium citrate, sodium phosphate and magnesium chloride, and may also include mannitol and/or glucose.
In one embodiment, the synthetic media is contained in a first blood bag and said platelet preparation is contained in a second blood bag, the synthetic media being added to the platelet preparation in step (b) by expressing the synthetic media from the first blood bag into the second blood bag via a sterile connection.
In a preferred embodiment, the compound is either 5xe2x80x2-(4-amino-2-oxa)butyl-4,4xe2x80x2,8-trimethylpsoralen or 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen.
In one embodiment, the method described above includes administering said platelet preparation by intravenous infusion to a mammal.
The present invention also contemplates a method of synthesizing 4,8-dialkyl-5xe2x80x2-bromomethyl-4xe2x80x2-methylpsoralens, without performing chloromethylation, comprising: a) providing 4,8-dialkyl-7-(1-methyl-2-oxopropyloxy)psoralen; d) stirring 4,8-dialkyl-4xe2x80x2,5xe2x80x2-dimethylpsoralen in carbon tetrachloride to obtain 4,8-dialkyl-5-bromomethyl-4xe2x80x2-methylpsoralen. A method of synthesizing 4,8-dialkyl-4xe2x80x2-bromomethyl-5xe2x80x2-methylpsoralens, without performing chloromethylation, is contemplated, comprising: a) providing 4,8-dialkyl-7-(1-methyl-2-oxopropyloxy)psoralen; d) stirring 4,8-dialkyl-4xe2x80x2,5xe2x80x2-dimethylpsoralen in methylene chloride to obtain 4,8-dialkyl-4xe2x80x2-bromomethyl-5xe2x80x2-methylpsoralen.
A novel compound is also contemplated, having the formula: 
or a salt thereof.
Finally, the present invention contemplates compositions having anti-viral properties. The first comprising an aqueous solution of a 4xe2x80x2-primaryamino-substituted psoralen and platelets suitable for in vivo use. One embodiment, further comprises a synthetic media, comprising sodium acetate, potassium chloride, sodium chloride, sodium citrate, sodium phosphate and magnesium chloride and optionally mannitol or glucose. These same compositions are contemplated that contain a 5xe2x80x2-primaryamino-substituted psoralen rather than a 4xe2x80x2-primaryamino-substituted psoralen.
A novel synthetic platelet storage media, is also contemplated, comprising an aqueous solution of:
45-100 mM sodium chloride;
4-5 mM potassium chloride;
10-15 mM sodium citrate;
20-27 mM sodium acetate;
0-2 mM glucose;
0-30 mM mannitol;
approximately 20 mM sodium phosphate;
2-3 mM magnesium chloride; and
a psoralen selected from the group consisting of 4xe2x80x2-primaryaminopsoralen and a
5xe2x80x2-primaryaminopsoralen, at a concentration between approximately 0.1 and 250 xcexcM.
The present invention provides a method of inactivating nucleic acid-containing pathogens in blood products, comprising providing, in any order, psoralen, photoactivation means, a blood product intended for in vivo use suspected of being contaminated with at least one pathogen, adding psoralen to the blood product to create a solution of psoralen at a concentration, treating the solution with photoactivation means so as to create a treated blood product, wherein pathogens are inactivated, and wherein at least a portion of the psoralen concentration is free in solution; and removing substantially all of the portion of psoralen concentration free in solution in treated blood product. In one embodiment, the removing step comprises contacting treated blood product with a resin. It is contemplated that various resins will be used with the present invention, including but not limited to adsorbents, polystyrene, polyacrylic ester, silica, activated charcoal, and activated charcoal coated with poly-(2-hydroxyethyl methacrylate). In an alternative embodiment, the contacting step comprises perfusing blood product through an in-line column containing resin.
In another embodiment, the method of the present invention comprises passing blood product through a flow adapter in fluidic contact with an in-line column after the blood product has passed through the in-line column. In another embodiment the contacting occurs within a bag containing resin. In a particularly preferred embodiment, the resin is contained within a mesh enclosure in the bag, wherein the mesh enclosure is adapted to allow blood product to contact the resin.
In another embodiment, the method of the present invention further comprises a partition mounted external to, and in contact, with the bag, wherein the partition is adapted to separate blood product from the mesh enclosure and adapted to be removed from the bag at a predetermined time. In an alternative embodiment, the method further comprises mixing the resin-containing bag with a shaker device. It is contemplated that various psoralen compounds will be useful in the present invention, including, but not limited to 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen. It is also contemplated that the blood product comprise any blood components, including but not limited to platelets, plasma, red cells, and white cells, as well as whole blood.
In another embodiment, the present invention provides a method of inactivating nucleic acid-containing pathogens in blood products, comprising the steps of, providing in any order, 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen, photoactivation means, a platelet mixture intended for in vivo use suspected of being contaminated with pathogens, adding 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen to the platelet mixture to create a solution of 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen at a concentration; treating the solution with photoactivation means so as to create a treated platelet mixture wherein pathogens are inactivated and wherein at least a portion of 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen concentration is free in solution; and removing substantially all of the portion of 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen concentration free in solution in the treated platelet mixture.
In one embodiment of this method, the removing step comprises contacting treated platelet mixture with a resin. The present invention contemplates greater than 99% removal of 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen at two hours with contacting with a resin. It is contemplated that various resins will be used with the present invention, including but not limited to adsorbents, polystyrene, polyacrylic ester, silica, activated charcoal, and activated charcoal coated with poly-(2-hydroxyethyl methacrylate). In an alternative embodiment, the contacting step comprises perfusing blood product through an in-line column containing resin. In yet another embodiment, this method further comprises passing blood product through a flow adapter in fluidic contact with an in-line column after blood product has passed through the in-line column.
In one embodiment of this method, the contacting occurs within a bag containing resin. In a preferred embodiment, the resin is contained within a mesh enclosure in the bag, wherein the mesh enclosure is adapted to allow blood product to contact resin. In another preferred embodiment, the method further comprises a partition mounted external to, and in contact, with the bag, wherein the partition is adapted to separate the blood product from the mesh enclosure and adapted to be removed from the bag at a predetermined time. It is contemplated that this method further comprises mixing the resin-containing bag with a shaker device.
The present invention also provides a blood decontamination system, comprising a first blood bag and an in-line column containing resin capable of removing psoralen, where the in-line column has an input end in fluidic communication with first blood bag, an output end, and a capacity. In one embodiment, the output end is in fluidic contact with a second blood bag. In a preferred embodiment, the capacity of the in-line column is approximately 5-10 mL. In another embodiment, the method further comprises a flow adapter positioned in fluidic contact with the in-line column and positioned after the output end of the inline column and before the second bag.
In one embodiment of this method, the removing step comprises contacting treated platelet mixture with a resin. It is contemplated that various resins will be used with the present invention, including but not limited to adsorbents, polystyrene, polyacrylic ester, silica, activated charcoal, and activated charcoal coated with poly-(2-hydroxyethyl methacrylate). In an alternative embodiment, the contacting step comprises perfusing blood product through an in-line column containing resin. In yet another embodiment, this method further comprises passing blood product through a flow adapter in fluidic contact with an in-line column after blood product has passed through the in-line column.
The present invention also provides a blood bag, comprising a biocompatible housing and a compartment within the housing which contains a resin capable of removing psoralen. In one embodiment, the blood bag further comprises a mesh enclosure disposed within the compartment and containing resin, wherein the mesh enclosure is adapted to allow a blood product to contact the resin. It is contemplated that the mesh enclosure is fixed in location within the compartment.
In an alternative embodiment, the blood bag further comprises a partition mounted external to, and in contact with, the biocompatible housing, wherein the partition is adapted to separate blood product from the mesh enclosure and to be removed from the bag at a predetermined time to allow blood product to contact the resin. In yet another embodiment, the blood bag further comprises a flow adapter in fluidic contact with the biocompatible housing and having a 50-100 xcexcm opening mesh filter. It is contemplated that the resin of this invention comprise various materials, including, but not limited to adsorbents, polystyrene, polyacrylic ester, silica, activated charcoal, and activated charcoal coated with poly-(2-hydroxyethyl methacrylate).
It is contemplated that various blood bags will be used. It is not intended that the blood bag be limited to a particular type or source. Indeed, it is contemplated that blood bags obtained from any commercial source will be useful in the present invention. Also, it is contemplated that the photoactivation device of the present invention may be obtained from any commercial source. Thus, it is not intended that the present invention be limited to any one source of blood bag or photoactivation device.
The present invention contemplates a container for a blood product, comprising: a) a biocompatible housing; b) a resin capable of removing psoralen from the blood product, the resin contained within the biocompatible housing; and c) means for retaining the resin within the biocompatible housing.
The present invention also contemplates a blood bag, comprising: a) a biocompatible housing; b) a resin capable of removing aminopsoralen from a blood product, the resin contained within the biocompatible housing; c) means for retaning the resin within the biocompatible housing.
In some embodiments, the retaining means of the container or the blood bag comprises a mesh enclosure disposed within the biocompatible housing, the mesh enclosure containing the resin and adapted to allow a blood product to contact the resin. In further embodiments, the mesh enclosure comprises 30 xcexcm pores. In particular embodiments, the mesh enclosure comprises polyester.
In additional embodiments, the container or the blood bag further comprises an inlet/outlet line. In still further embodiments, the retaining means comprises a mesh filter positioned in the inlet/oulet line and in fluidic communication with the biocompatible housing. The mesh filter comprises 30 xcexcm pores in particular embodiments, while the mesh of the mesh filter comprises polyester in still other embodiments.
In particular embodiments of the present invention, the resin is adsorbent. When the resin is adsorbent it comprises a polymer in some embodiments. The polymer may be polystyrene in additional embodiments, and the polystyrene is crosslinked in still further embodiments.
In certain embodiments, the aminopsoralen is 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen.
The present invention also contemplates a method of inactivating nucleic acid-containing pathogens in blood products, comprising: a) providing, in any order: i) psoralen, ii) photoactivation means, iii) a first container containing a blood product intended for in vivo use suspected of being contaminated with the pathogens; b) adding the psoralen to the blood product in the first container to create a solution of psoralen at a concentration; c) treating the solution with the photoactivation means so as to create a treated blood product wherein the pathogens are inactivated and wherein at least a portion of the psoralen concentration is free in the solution; and d) removing some of the portion of the psoralen free in solution in the treated blood product. It should be emphasized that the present invention is not limited to the removal of a particular amount of psoralen free in solution. Indeed, the present invention contemplates the removal of any portion of psoralen free in solution.
In particular embodiments, the psoralen is 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen. In other embodiments, the psoralen is brominated. When a brominated psoralen is used, the brominated psoralen may be 5-bromo-8-methoxypsoralen or 5-bromo-8-(diethylaminopropyloxy)-psoralen. Moreover, the psoralen is a quaternary amine in some embodiments, and the quaternary amine psoralen is 4xe2x80x2-(triethylamino) methyl-4,5xe2x80x2,8-trimethylpsoralen in still further embodiments.
In some embodiments of the present invention, the removing step comprises transferring the treated blood product into a second container, comprising: i) a biocompatible housing; ii) a resin capable of removing psoralen from the blood product, the resin contained within the biocompatible housing; and iii) retaining means for retaining the resin within the biocompatible housing under conditions such that some of the portion of the psoralen concentration free in solution is removed in the treated blood product.
In some embodiments, the retaining means of the container or the blood bag comprises a mesh enclosure disposed within the biocompatible housing, the mesh enclosure containing the resin and adapted to allow a blood product to contact the resin. In further embodiments, the mesh enclosure comprises 30 xcexcm pores. In particular embodiments, the mesh enclosure comprises polyester.
In additional embodiments, the container or the blood bag further comprises an inlet/outlet line. In still further embodiments, the retaining means comprises a mesh filter positioned in the inlet/oulet line and in fluidic communication with the biocompatible housing. The mesh filter comprises 30 xcexcm pores in particular embodiments, while the mesh of the mesh filter comprises polyester in still other embodiments.
The present invention also contemplates a method of inactivating nucleic acid-containing pathogens in blood products, comprising: a) providing, in any order: i) a donor, the donor capable of providing blood suspected of being contaminated with the pathogens, ii) blood separation means for separating the blood into blood products, iii) psoralen, iv) photoactivation means, and v) psoralen removal means; b) withdrawing the blood from the donor and introducing blood into said blood separation means; c) isolating a blood product from the blood with the blood separation means; d) adding the psoralen to the blood product to create a solution of psoralen at a concentration; e) treating the solution with the photoactivation means so as to create a treated blood product wherein the pathogens are inactivated and wherein at least a portion of the psoralen concentration is free in the solution; and f) removing substantially all of the portion of the psoralen free in solution in the treated blood product with the psoralen removal means.
In particular embodiments, the blood separation means is an apheresis system. The blood product is platelets in certain embodiments, and plasma in other embodiments.
In some embodiments, the psoralen removal means comprises a mesh enclosure containing a resin, the mesh enclosure adapted to allow a blood product to contact the resin. The resin is adsorbent in some embodiments. When the resin is adsorbent, it may be a polymer in further embodiments. In particular embodiments, the polymer comprises polystyrene, while the polystyrene is crosslinked in still further embodiments.
The psoralen may be an aminopsoralen in some embodiments, and a brominated psoralen in others.
Additionally, the present invention contemplates a method of inactivating nucleic acid-containing pathogens in blood products, comprising: a) providing, in any order: i) a donor, the donor capable of providing blood suspected of being contaminated with the pathogens, ii) an apheresis system for separating platelets from the blood, iii) an aminopsoralen, iv) photoactivation means, and v) psoralen removal means; b) withdrawing the blood from the donor and introducing the blood into the apheresis system; c) isolating the platelets from the blood with the apheresis system; d) producing a platelet mixture comprising the platelets; e) adding the aminopsoralen to the platelet mixture to create a solution of aminopsoralen at a concentration; f) treating the solution with the photoactivation means so as to create a treated platelet mixture wherein the pathogens are inactivated and wherein at least a portion of the aminopsoralen concentration is free in the solution; and g) removing substantially all of the portion of the aminopsoralen free in solution in the treated platelet mixture with the psoralen removal means.
In some embodiments, the psoralen removal means comprises a mesh enclosure containing the resin, the mesh enclosure adapted to allow a platelet mixture to contact the resin. The resin is adsorbent in some embodiments. When the resin is adsorbent, it may be a polymer in further embodiments. In particular embodiments, the polymer comprises polystyrene, while the polystyrene is crosslinked in still further embodiments. Finally, the resin is subjected to a wetting process in still additional embodiments.
In still further embodiments, the aminopsoralen is 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen.
The present invention also contemplates a method of inactivating nucleic acid-containing pathogens in blood products, comprising: a) providing, in any order: i) a donor, the donor capable of providing blood suspected of being contaminated with the pathogens, ii) an apheresis system for separating platelets from the blood, iii) synthetic media, iv) a platelet collection container, v) 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen, vi) photoactivation means, and vii) psoralen removal means; b) withdrawing the blood from the donor and introducing the blood into the apheresis system; c) isolating the platelets from the blood with the apheresis system; d) collecting the platelets in a platelet container over a period of time; e) adding the synthetic media to the platelets in the platelet container, thereby producing a platelet mixture comprising platelets and synthetic media; f) adding the 4xe2x80x2-(4-amino-2oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen to the platelet mixture to create a solution of 4xe2x80x2-(4amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen at a concentration; g) treating the solution with the photoactivation means so as to create a treated platelet mixture wherein the pathogens are inactivated and wherein at least a portion of the 4xe2x80x2-(4-amino-2oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen concentration is free in the solution; and h) removing substantially all of the portion of the 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8trimethylpsoralen free in solution in the treated platelet mixture with the psoralen removal means.
In some embodiments, the synthetic media comprises phosphate. In still further embodiments, the synthetic media is added to the platelets over the period of time the platelets are being collected.
In some embodiments, the psoralen removal means comprises a mesh enclosure containing the resin, the mesh enclosure adapted to allow a platelet mixture to contact the resin. The resin is adsorbent in some embodiments. When the resin is adsorbent, it may be a polymer in further embodiments. In particular embodiments, the polymer comprises polystyrene, while the polystyrene is crosslinked in still further embodiments. Finally, the resin is subjected to a wetting process in still additional embodiments.
The term xe2x80x9cblood productxe2x80x9d refers to all formulations of the fluid and/or associated cellular elements and the like (such as erythrocytes, leukocytes, platelets, etc.) that pass through the body""s circulatory system; blood products include, but are not limited to, platelet mixtures, serum, and plasma. The term xe2x80x9cplatelet mixturexe2x80x9d refers to one type of blood product wherein the cellular element is primarily or only platelets. A platelet concentrate (PC) is one type of platelet mixture where the platelets are associated with a smaller than normal portion of plasma. A synthetic media may make up that volume normally occupied by plasma; for example, a platelet concentrate may entail platelets suspended in 35% plasma/65% synthetic media. Frequently, the synthetic media comprises phosphate.
The term xe2x80x9cphotoproductxe2x80x9d refers to products that result from the photochemical reaction that a psoralen undergoes upon exposure to ultraviolet radiation.
The term xe2x80x9cresinxe2x80x9d refers to a solid support (such as beads/particles etc.) capable of interacting and attaching to various elements, including psoralens, in a solution or fluid (e.g., a blood product), thereby removing those elements. The removal process is not limited to any particular mechanism. For example, a psoralen may be removed by an adsorbent or by charge (i.e., affinity interaction). The term xe2x80x9cadsorbent resinxe2x80x9d refers broadly to both natural organic substances and synthetic substances. Various adsorbent resins differ in surface area, pore size, chemical nature (e.g., polystyrene divinylbenzene and acrylic ester), polarity, etc., to allow optimum performance for particular applications (e.g., adsorption of pharmaceuticals). The adsorbent resins may be packaged in a number of arrangements, including a column through which a substance like blood can be perfused, and a mesh having apertures sized to allow contact of the adsorbent with the substance while retaining the adsorbent resin within the area defined by the mesh.
The term xe2x80x9cpsoralen removal meansxe2x80x9d refers to a substance or device that is able to remove psoralen from, e.g., a blood product. A psoralen removal means may also remove other components of the blood product, such as psoralen photoproducts. A preferred psoralen removal means is an adsorbent resin.
The term xe2x80x9cin-line columnxe2x80x9d refers to a container, usually cylindrically shaped, having an input end and an output end and containing a substance disposed therein to remove substances from a blood product. The present invention contemplates the use of a column having a capacity of at least 1 mL, and preferably 5-10 mL that is packed with an adsorbent resin for removing psoralens and psoralen photoproducts from the blood product. A blood product enters the input end, comes in contact with the adsorbent resin, and then exits the output end.
The term xe2x80x9cpartitionxe2x80x9d refers to any type of device or element that can separate or divide a whole into sections or parts. For example, the present invention contemplates the use of a partition to divide a blood bag, adapted to contain a blood product, into two parts. The blood product occupies one part of the bag prior to and during treatment, while the adsorbent resin occupies the other part. In one embodiment, after treatment of the blood product, the partition is removed (e.g., the integrity of the partition is altered), thereby allowing the treated blood product to come in contact with the adsorbent resin. The partition may either be positioned in the bag""s interior or on its exterior. When used with the term xe2x80x9cpartition,xe2x80x9d the term xe2x80x9cremovedxe2x80x9d means that the isolation of the two parts of the blood bag no longer exists; it does not necessarily mean that the partition is no longer associated with the bag in some way.
The term xe2x80x9cflow adapterxe2x80x9d refers to a device that is capable of controlling the flow of a particular substance like a blood product. The flow adapter may perform additional functions, such as preventing the passage of pieces of adsorbent resin material.
The term xe2x80x9cresin retaining meansxe2x80x9d refers to any mechanism that confines resin to a defined area, like a biocompatible housing. For example, a mesh enclosure, housed within a platelet storage container, may be used to hold the resin within the container. Similarly, a filter (e.g., a mesh filter) may be positioned at the inlet/outlet line of a blood product storage bag. The term xe2x80x9cinlet/outlet linexe2x80x9d refers to the tubing that is connected to and in fluidic communication with a blood product storage bag. There may be a single inlet/outlet line or two or more lines connected to a bag.
The terms xe2x80x9cmesh enclosure,xe2x80x9d xe2x80x9cmesh pouchxe2x80x9d and the like refer to an enclosure, pouch, bag or the like manufactured to contain multiple pores. For example, the present invention contemplates a pouch, containing the adsorbent resin, with pores of a size that allow a blood product to contact the adsorbent resin, but retain the resin within the pouch. For purposes of the present invention, the adsorbent-containing mesh enclosure is referred to as a RD. In a preferred embodiment, the RD is housed in a blood product storage container (e.g., a platelet storage container). The present invention contemplates that mesh enclosures will be constructed of a woven, medical-grade polyester mesh or other suitable material like nylon. The preferred range of pore size of the mesh material is approximately 10 xcexcm and 50 xcexcm, while the preferred embodiment of the present invention utilizes mesh with pores of approximately 30 xcexcm.
The terms xe2x80x9cfluidic contact,xe2x80x9d xe2x80x9cfluidic connection,xe2x80x9d and the like refer to the ability of a fluid component (e.g., a blood product) to flow from one element to another. For example, a blood component may flow from a platelet bag through tubing to a flow adapter; thus, the flow adapter does not have to be in direct contact with the platelet bag. Similarly, tubing from each of two or more blood product containers may be connected (e.g., sterile welded) using a sterile connection device to allow fluid to be transferred from one container to another.
The phrase xe2x80x9cadapted to allow a blood product to contact said resinxe2x80x9d refers to the ability of a blood product to contact and interact with a resin such that the resin is able to adsorb components (e.g., psoralen and psoralen photoproducts) from the blood product. The phrase is frequently used to describe the ability of a psoralen- and irradiation-treated blood product (e.g., platelets), contained within a blood product storage container, to pass through the pores of a mesh enclosure housed within that container; in so doing, the resin is able to adsorb the psoralen and psoralen photoproducts.
The term xe2x80x9cshaker devicexe2x80x9d refers to any type of device capable of thoroughly mixing a blood product like a platelet concentrate. The device may have a timing mechanism to allow mixing to be restricted to a particular duration.
The term xe2x80x9cbiocompatible housingxe2x80x9d refers broadly to housings, containers, bags, vessels, receptacles, and the like that are suitable for containing a biological material, such as whole blood, platelet concentrates and plasma. Suitable containers are biocompatible if they have minimal, if any, effect on the biological material to be contained therein. By xe2x80x9cminimalxe2x80x9d effect it is meant that no significant difference is seen compared to the control. Thus, blood products may be stored in biocompatible housings prior to transfusion to a recipient. In a preferred embodiment, a biocompatible housing is a platelet storage container.
The term xe2x80x9cblood separation meansxe2x80x9d refers broadly to a device, machine, or the like that is able to separate blood into blood products (e.g., platelets and plasma). An apheresis system is one type of blood separation means. Apheresis systems generally comprise a blood separation device, an intricate network of tubing and filters, collection bags, an anticoagulant, and a computerized means of controlling all of the components. The blood separation device is most commonly a centrifuge. At least one pump is used to move the blood, separated blood components, and fluid additives through the apheresis system and ultimately back to either the donor or to a collection bag(s). Though not limited to any particular type of apheresis system, the present invention specifically contemplates the use of automated systems that are capable of collecting a particular amount of a desired blood product mixture.
The term xe2x80x9cisolatingxe2x80x9d refers to separating a substance out of a mixture containing more than one component For example, platelets may be separated from whole blood. The product that is isolated (e.g, platelets) does not necessarily refer to the complete separation of that product from other components. For example, platelets isolated by an apheresis system frequently are associated with a small volume of plasma; in this example, the platelets would still be deemed to have been separated from the whole blood.
The term xe2x80x9cfilterxe2x80x9d refers broadly to devices, materials, and the like that are able to allow certain components to a mixture to pass through while retaining other components. For example, a filter may comprise a mesh with pores sized to allow a blood product (e.g., plasma) to pass through, while retaining other components such as resin particles. The term xe2x80x9cfilterxe2x80x9d is not limited to the means by which certain components are retained.
The term xe2x80x9cpolyesterxe2x80x9d refers broadly to materials comprising [poly(ethylene terephthalate)]. The polyester material may be in the form of a mesh material with pores of a definitive size.
The term xe2x80x9cpolymerxe2x80x9d refers broadly to a material made up of a chain of identical, repeated xe2x80x9cbase unitsxe2x80x9d. The term encompasses materials containing styrene (C6H5CHxe2x95x90CH2) monomers, which may be referred to as xe2x80x9cpolystyrene networks.xe2x80x9d
The term xe2x80x9ccrosslinkedxe2x80x9d refers broadly to linear molecules that are attached to each other to form a two- or three-dimensional network. For example, divinylbenzene (DVB) serves as the crosslinking agent in the formation of styrene-divinylbenzene copolymers. The term also encompasses xe2x80x9chypercrosslinkingxe2x80x9d in which hypercrosslinked networks are produced by crosslinking linear polystyrene chains either in solution or in a swollen state with bifunctional agents (described below).
The terms xe2x80x9caminopsoralenxe2x80x9d xe2x80x9caminated psoralenxe2x80x9d and the like refer to psoralen compounds that contain an NH2 group linked to either the 4xe2x80x2-position (4xe2x80x2-primaryamino-substituted psoralens) or the 5xe2x80x2-position (5-primaryamino-substituted psoralens) of the psoralen by a hydrocarbon chain. In 4xe2x80x2-primaryamino-substituted psoralens, the total length of the hydrocarbon chain is 2-20 carbons, where 0 to 6 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon. 4xe2x80x2-primaryamino-substituted psoralens may have additional substitutions on the 4, 5xe2x80x2, and 8 positions of the psoralen, said substitutions include, but are not limited to, the following groups: H and (CH2)nCH3, where n=0-6. By comparison, in 5xe2x80x2-primaryamino-substituted psoralens, the total length of the hydrocarbon chain is 1-20 carbons, where 0 to 6 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon. 5xe2x80x2-primaryamino-substituted psoralens may have additional substitutions on the 4, 4xe2x80x2, and 8 positions of the psoralen, said substitutions include, but are not limited to, the following groups: H and (CH2)nCH3, where n=0-6.
The term xe2x80x9cbrominated psoralenxe2x80x9d refers to psoralen compounds that contain a bromine (Br) atom linked thereto. Preferred brominated psoralens contain a bromine linked to the 5-position. Examples of brominated psoralens included 5-bromo-8methoxypsoralen and 5-bromo-8-(diethylaminopropyloxy)-psoralen.